Method for generating overexpression of alleles in genes of unknown function

ABSTRACT

Methods for generating and using novel overexpression activity alleles of a gene in any organism, especially  Drosophilia , are provided. Such alleles may be utilized in screening assays, and used to generate dominant-negative forms of bacterial toxins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/263,367 filed Oct. 1, 2002, now issued as U.S. Pat. No.7,193,126; which claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 60/326,546, filed Oct. 1, 2001, now abandoned. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

GRANT INFORMATION

This work was supported by National Institutes of Health # NS29870 andNSF # NSFIBN-9604048.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally concerns procedures for generatingtransgenic flies for expression and study of a gene of interest. Theinvention particularly concerns processes for generating and usingstocks of transgenic Drosophila that carry a mutant allele of a gene ofunknown function related to the production of a new dominant phenotype,as well as methods for utilizing transgenic Drosophila to generate andstudy other genes, such as those encoding dominant-negative bacterialtoxins.

2. Description of Related Art

Pharmacological research is hampered by the labor-intensive andextremely lengthy identification and systematic characterizationprocedures for new therapeutic compounds. A conventional processinvolves the screening of Thousands of individual compounds there areconventionally screened for a desired biological therapeutic benefit.Less than 1 in 10,000 of the synthetic compounds screened haveultimately been approved by the Food and Drug Administration. The costapproximates $200+ million per drug put into service. Natural productshave provided the impetus for search of therapeutically effectivepharmacological compounds for many years. Complex mixtures derived fromcells, or their metabolites, are screened for biological activity, andthe specific molecule possessing the activity is purified using thebiological activity as the means for identifying the component of themixture possessing the desired activity.

An alternative methodology has been to screen previously synthesizedindividual compounds saved in “libraries” in drug companies or researchinstitutions. More recently, peptide or oligonucleotide libraries havebeen developed which may be screened for a specific biological function.

Because many of the existing therapeutics have been identifiedaccidentally, their mechanism of action is not well understood. A moreeffective approach to identifying new molecular compounds effectiveagainst various disease conditions requires precise knowledge of themolecular defect underlying a given disease, and of the cellularpathways and processes of action. This is a weakness inherent in thecurrent screening methodologies of compound libraries employingcell-free and in vitro cell-based assays.

It should also be noted that high throughput screening does not actuallyidentify a drug, but merely high-quality “hits” or “leads” that areactive at a relatively low concentration. This powerful screening toolalso suffers from a number of limitations like bioavailability, toxicityand specificity. Subsequent studies are required before such a compoundmetamorphosizes into a therapeutically useful drug.

Screening of compound libraries with cell-free and in vitro assaysystems has intrinsic limitations and weaknesses. For example, incell-free systems, screening is limited to single target molecules, andthey do not provide an inherent test for either specificity of theinteraction, or of the toxicity of the test compound. But, perhaps mostimportantly, cell-free systems do not identify intermediary targets insignaling pathways made up of a hodge-podge of cell membrane,cytoplasmic, and nuclear-based components not present in cell-freesystems.

Cell-based assays have several advantages over cell-free systems, thefirst being the capacity of self-replication. Moreover, the interactionsoccur in a biological context hopefully more closely mimicking thenormal physiological conditions in vivo. Bioavailability andcytotoxicity are more easily assessed, but they often provide inadequatesimilarity to the in vivo disease conditions developing in multicellulartissues.

With the current screens using cell-free or in vitro cell-based assays,these differentiation end-points are difficult to assess and cell numberor cell mass may be the more appropriate assay for their high-throughputdesigns. This is due to the fact that current screening methodologiescan not easily discriminate growth arrest due to differentiation fromother antiproliferative or simple cytotoxic effects.

Whole embryo cultures have also been used to screen for chemical effectsin, for example, rodents and chickens. Adverse embryonic outcomes(malformations or embryotoxicity) are directly related to the serumconcentration of the compound being tested. These serum concentrationscan be directly compared to the serum concentration in the human. Wholeembryo culture systems are problematic in that they result in largenumbers of in vivo false-positives, and development within the culturesis limited to the very early stages of embryogenesis.

Similarly, the nematode C. elegans is frequently utilized as a modelorganism for the genetic dissection of developmental controls and cellsignaling. However, in C. elegans there are no genetically sensitizedsystems available that permit reliable detection of even a two-foldreduction in a signaling process caused by either a chemical compound ora mutation in a gene. Although C. elegans can be grown in microtiterplates, the phenotypic screens are markedly limited. Also, chemicalcompounds would necessarily be administered by feeding, and would thuspossess all of the aforementioned inherent disadvantages.

Another widely-utilized model genetic system is yeast. Although yeastare easily maintained and can readily be grown in large numbers, theyare a simple, single-celled organism and thus possess the inherentlimitation of being incapable of replicating a complex, multi-cellularsystem. Although the yeast system offers a comparatively higherthroughput, it possesses inherent limitations, as most diseaseconditions are dependent upon cell-cell interactions within tissues thatcannot be modeled in yeast. Finally, and most importantly, the overalldegree of conservation of signaling pathways between yeast and human issignificantly lower than that between Drosophila and humans.

Studies in the fruit fly Drosophila melanogaster have altered ourestimate of the evolutionary relationship between vertebrate andinvertebrate organisms. Key molecular pathways required for thedevelopment of a complex animal, such as patterning of the primary bodyaxes, organogenesis, wiring of a complex nervous system and control ofcell proliferation have been highly conserved since the evolutionarydivergence of flies and humans. When these pathways are disrupted ineither vertebrates or invertebrates, similar defects are often observed.The utility of Drosophila as a model organism for the study of humangenetic disease is now well documented. Developmental defects such asthe mesenchymal malformations associated with Saethre-Chotzen syndrome(Howard et al. 1997), formation of intracellular inclusions inpolyglutamine-tract repeat disorders such as spinocerebellar ataxia andHuntington disease (Fortini and Bonini 2000), and loss ofcellular-growth control and malignancy resulting from mutations of tumorsuppressor genes (Potter et al. 2000) have been analyzed effectivelyusing Drosophila as the model genetic system. The many basic processesthat are shared between Drosophila and humans, in conjunction with therecent completion of the Drosophila genomic sequence, provide thenecessary ingredients for launching systematic analyses of humandisease-causing genes in Drosophila.

The value of Drosophila as a screening system for evaluating thebiological activities of chemicals has been well documented (see e.g.,Schulz, et al, 1955. Cancer Res. 3(suppl.): 86-100; Schuler, et al.,1982. Terat. Carcin. Mutag. 2:293-301). Small numbers of chemicalsubstances are administered to larvae or flies by feeding, and flies arethen analyzed for survival and for phenotypic alterations. Althoughthese conventional tests show the potential use of Drosophila as a toolto analyze the function of small molecular weight compounds, thesemethods neither permit high-throughput screens, nor permit the directedsearch for small molecular weight compounds that interfere with aspecific morphogenetic pathway related to a human disease condition.Application of compounds by feeding requires relatively large amounts ofthe substance, and its uptake by the larvae and thus its finalconcentration is, at best, difficult to control. Furthermore,application by feeding does not permit automation of the procedurenecessary for high-throughput analysis (Ernst Hafen, United StatesPatent Application, 20020026648).

The Drosophila epidermal growth factor (EGF)-receptor tyrosinekinase(EGF-R) controls a large array of cell-fate choices throughout the lifecycle (Schweitzer, R. & Shilo, B. Z. (1997), Perrimon, N. & Perkins, L.A. (1997)) and can be activated by multiple ligands (Moghal & Sternberg(1999)). Among them, Vein is directly secreted to signal to adjacentcells (Schnepp, et al (1996)), whereas other EGF ligands such as Spitz(Spi) (Rutledge, et al (1992)) and Gurken (Grk) (Neuman-Silberberg &Schupbach (1993)) are similar to the human transforming growth factor aand are initially expressed as membrane-bound precursors. Numerousstudies have provided corroborating evidence that these latter precursorligands are initially inert and depend on two accessory membraneproteins, Rhomboid (Rohr) and Star, to be processed into activediffusible forms (Schweitzer et al (1995); Guichard Et Al (1999); Pickup& Banerjee (1999); Guichard Et Al (1999); Bang & Kintner (2006);Guichard Et Al (2000); Lee Et Al (2001); Urban Et Al (2001); Golembo EtAl (1996)). Rho is a predicted seven-pass transmembrane protein (Bier etal (1990)), and Star is predicted to be a type II single-passtransmembrane protein predominantly localized in the endoplasmicreticulum (ER) (Pickup & Banerjee (1996); Kolodkin et al (1994)), whichacts as an obligate partner of Rho to activate EGF-R signaling in a cellnonautonomous fashion (Bier et al (1990)). Recent studies show that Staris necessary for Spi to translocate from the ER to the Golgi apparatus,where it is directly cleaved by Rho, a novel type of intramembraneserine protease (Lee Et Al (2001); Urban Et Al (2001); Tsruya et al(2002)). Unlike the Egf-r, spitz (spi), and Star genes, which areexpressed ubiquitously in most epidermal cells, rhomboid (rho) isexpressed in a highly localized and dynamic pattern (Bier et al (1990))that correlates with the in situ activation pattern of mitogen-activatedprotein kinase (MAPK), an essential downstream component of all tyrosinekinase receptors (Gabay et al (1997) Science 277; Gabay et al (1997)Development 124; Bier (1998)). This latter observation suggests that Rhoprovides the appropriate restricted spatial and temporal activation formembrane-bound EGF ligands. A good example of the localized activity ofRho is provided by the wing disc, in which the restricted expression ofrho in longitudinal stripes controls the commitment of these cells tothe vein fate through the activation of EGF-RMAPK signaling. Thus, rhovemutants, who fail to express rho in vein primordia, lack sections ofveins, whereas ubiquitous ectopic expression of rho converts the entirewing blade into a single solid vein (Bang & Kintner (2000); Lindsley, D.& Zimm (1992); Sturtevant et al (1993)).

Current methods for generating gain-of-function mutations are of twogeneral sorts.

1) Structure/function studies in which mutant forms of a gene-x arecreated in vitro by one of a several of existing methods for making sitedirected mutations and then introduced into an organism to assay thefunction of the mutated gene.

2) Systematic screens for mutant alleles of the endogenous gene-x usingone of a variety of mutagens.

These two types of analysis are typically very labor intensive and canonly recover rather limited numbers of mutations. For example, in thecase of Inventors' structure/function analysis of the Drosophila soggene, one person spent approximately two years generating a collectionof 23 mutant forms of the gene, which were then transformed into fliesto obtain several independent transgenic lines of flies carrying eachconstruct. These mutant sog constructs were then misexpressed in thewing to test for the function of the mutated genes. Using this approachInventors identified two activities of Sog which had not been previouslyknown. Thus, whereas misexpression of wild-type sog during wingdevelopment causes a mild loss-of vein phenotype (FIG. 1C; Yu et al.,1996), misexpression of one mutant truncated form of sog—referred to assupersog—generates more severe wing patterning defects (FIG. 1D; Yu etal., 2000), while misexpression of a second truncated form inducesproduction of ectopic wing veins (Yu et al., manuscript in preparation).Inventors and other investigators have also conducted several differentsystematic screens for mutations in the endogenous sog gene, whichcumulatively amounted to at least one year of work by a single person.These tedious screens lead to the isolation of only null and partialloss-of-function sog alleles (Wieschaus et al., 1984; Ferguson et al.,1992; Francois et al., 1994).

What is needed is a method that could be employed to generate dominantalleles of a wide range of genes using various mutagens to provideinsight to the function and mechanism of action of novel genes. Thisapproach should be of particular utility in investigating the functionof human disease genes, which have no known functional motifs but havehomologues in Drosophila (Reiter et al (2001)). Furthermore, the methodshould be applicable to any organism in which it is possible tomisexpress transgenic constructs at high levels in a conditionalfashion.

SUMMARY OF THE INVENTION

The primary object according to this invention is to provide a geneticmethod for generating a novel overexpression activity allele(hereinafter “NOVA”) of a gene of interest. Novel overexpressionactivity alleles of a gene of unknown function can be powerful genetictools that can provide important insights into the function of thatgene.

Another object according to this invention resides in the ability to,once a NOVA allele of a gene has been isolated, design mutant screens toidentify second-site mutations in other genes acting in the same pathwayas the gene of interest. Mutants in these second-site modifier locialter the NOVA phenotype, either by enhancing or suppressing thatphenotype.

Still another object according to this invention is the potential use ofmutagens to revert NOVA mutant phenotypes generated by this method, andthereby efficiently generate second-site intragenic loss-of-functionalleles within the gene of interest for structure/function studies anddesigning therapeutics for human diseases.

In accordance with these objects, this invention contemplates a methodfor generating, in virtually any organism, a novel overexpressionactivity (NOVA) allele of a gene (gene-x) of unknown function. Themethod comprises misexpressing a wild-type copy of a gene by knownmethods, determining the phenotypic consequence of the misexpressing,namely transgene-x, and mutagenizing male organisms carrying thistransgene-x. The mutants, for example Drosophila males, are mated enmasse to relevant female strains, thereby activating global orrestricted expression. The progeny are screened for new NOVA dominantphenotypes resulting from the expression, and these NOVA mutant progenyare crossed to appropriate balancer females to establish stable stocks.The NOVA dominant phenotypes resulting from the expression can be inconstitutively active or dominant negative form.

It is also contemplated by this invention that any organism that can bemisexpressed and overexpressed is a candidate for this method. Mostpreferably, the organism is Drosophila. The mutagenizing contemplatedherein may be accomplished with chemical mutagens, for example, ethanemethyl sulfonate, an alkylating agent highly effective in eucaryoticsystems. The mutagenizing can also be accomplished with radiation, orany other factor capable of causing point mutation. Point mutations arepreferable because they allow precise targeting. They may be targeted atrandom sites in a particular region of DNA, or at a particular basepair.

The mutagenizing can also be accomplished with broadly functioningmutagens. Highly preferred methods contemplated by this invention causemutations enzymatically, preferably with the enzyme transposase. Theenzymic mutagenizing agent preferred in this invention is p-elementTransposase, most preferably, Δ2-3 Sb/TM6 Transposase.

Another, most preferred, embodiment in accordance with this invention isa method for generating novel mutations in human genes. Hereinagain, themethod involves the misexpressing of a wild-type copy of a gene by knownmethods, and determining the phenotypic consequence of themisexpressing, namely transgene-x. The method further involvesmutagenizing male flies carrying the transgene-x, activating global orrestricted expression by mating en masse to relevant female strains andscreening progeny for NOVA dominant phenotypes resulting from theexpression. The NOVA mutants are then crossed to appropriate balancerfemales to establish stable stocks. The misexpressed wild-type copy canbe one of approximately 1000 human disease genes currently identified inDrosophila.

A more specific and most preferred embodiment of this invention is amethod for generating dominant-negative forms of bacterial toxins usingNOVA screens in Drosophila, employing the following steps. The firstcontemplated step involves creating transgenic flies able to express abacterial toxin using the UAS/GAL4 conditional expression system. Thisis done by preparing DNA encoding full-length bacterial toxin (e.g. fromgenomic clone, plasmid subclone, or by PCR), inserting thetoxin-encoding gene into the pUAS-vector, transforming into E. ColiDH5α, and testing for successful recombinants. Next, the w+ markedpUAS-toxin DNA is purified and injected into w− fly embryos. F1generation w+ transformants are isolated and balanced, transformedpUAS-toxin lines are established. These lines are capable of expressingUAS-toxin in specific tissues, using a set of GAL lines to generateviable phenotypes (e.g. wingless or eyeless phenotypes).

The second contemplated step involves determining whether the Drosophilatoxin phenotype is caused by the same mechanism as in human cells byanalyzing the phenotype at the cellular level in third instar larvaecytoskeleton for toxins affecting Rho-like GTPases, For CytolethalDistending Toxins, this is preferably done by testing the reorganizationof actin to determine the shape and size of the cells. For LF of B.anthracis, determination is preferably accomplished by testing theactivity of MAPK, for example, using anti-diphosphoMAPK antibody.Preferably, testing the activity of Adenyl cyclase is also performed forEF of B. anthracis. All toxins are tested for capability of inducingcell-lethality, and whether said lethality occurs through apoptosis ornecrosis.

Preferably, genetic epistatsis experiments are used to confirm that thecellular targets of the toxin are fly homologues of the human targets.This is accomplished by testing to determine if the toxin-inducedphenotype is modified in a heterozygous mutant background for thepredicted targets and for their known partners, more preferablyscreening for enhancers of the toxin, and most preferably alsoperforming a phenotype analysis to identify such targets when thecellular target is not known.

The third contemplated step involves analyzing the effect of the noveldominant-negative toxins by establishing a mutant line carrying theUAS-toxinDN, determining what effect the toxinDN has when expressedalone, determining by PCR what lesion has been created in the UAS-toxininsertion and the corresponding altered protein sequence, and cloning amutant toxinDN gene into a fresh pUAS vector to obtain a construct. Theconstruct is transformed into flies and expressed in toxinDN gene flieswith GAL4. The final step is confirming that this is the only mutationrequired for DN toxin activity.

The advantages of the method according to this invention over existingmethods are the efficiency and speed with which it can be applied to awide variety of genes. Moreover, because the method is not predicated onassumptions based on protein domain structure and function, mutants canbe recovered in an unbiased fashion.

Still further objectives, embodiments and advantages of the inventionwill become apparent to those skilled in the art upon reading the entiredisclosure contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a genetic scheme for generating and screening for NOVA sogmutants. A) This crossing scheme is an example of a NOVA screen using afull length UAS-sog transgene as the target. Sog functions by inhibitingthe activity of BMP related ligands (François et al, 1994; Yu et al.,2000). B) A wild-type fly wing with the longitudinal veins (L1-L5) andthe anterior and posterior cross-veins (a-xv and p-xv respectively) areindicated. C) In previous studies (Yu et al., 1996; Yu et al., 2000)Inventors determined that misexpression of full length Sog throughoutthe wing primordium of developing larvae and pupae using a ubiquitousGAL4 source (MS 1096-GAL4=Ubi-GAL4) results in a very mild vein-lossphenotype in which cross-veins and distal tips of the L4 and L5 veinsare missing (compare to 1B). D) Misexpression of a truncated form of Sog(Supersog) causes a much more severe wing phenotype in which wing veinsfuse (e.g. L2 to L3 and L4 to L5), vein tissue is lost, and the wing issmaller in overall size (Yu et al., 2000).

FIG. 2 is a comparison of NOVA sog alleles with loss-of-functionmutations in gene encoding components of the BMP signaling pathway.Novel NOVA sog alleles isolated from the genetic scheme depicted in FIG.1 generate wing phenotypes when misexpressed during wing developmentthat are similar to those resulting from loss-of-function mutations ingene encoding components of the BMP signaling pathway. A) A mutant wingfrom an individual with reduced activity of the BMP ligand encoded bythe glass-bottom boat (gbb) gene. B) Ubiquitous misexpression of a fulllength UAS-sog transgene results in a phenotype very similar to that ofa loss-of-function gbb mutant (panel A). C) A mutant wing from anindividual with reduced activity of the BMP ligand encoded by thedecapentaplegic (dpp) gene in which wing veins fuse (e.g. L2 to L3 andL4 to L5). D) A wing misexpressing a truncated UAS-sog1 transgene(UAS-supersog1), which was generated by in vitro mutagenesis, in thesame cells that normally express the dpp gene. Note how similar thisphenotype is to that of a loss-of-function dpp mutant (panel C). E)Ubiquitous misexpression of the UAS-supersog1 transgene results in awing phenotype in which veins fuse (e.g. L2 to L3 and L4 to L5), veintissue is lost, and the wing is smaller in overall size. F) Ubiquitousmisexpression of the UAS-sog*5-73 mutant which was generated using thegenetic crossing scheme depicted in FIG. 1 results in a phenotype thatis nearly identical to that generated by the in vitro createdUAS-supersog1 (compare with panel E). G) A viable partial loss offunction mutant in the type-I BMP receptor encoded by the thick veins(tkv) gene results in thickened veins as a consequence of defects inlateral inhibitory signaling during pupal development. H) Ubiquitousmisexpression of the UAS-sog*5-8 mutant results in a thickened veinphenotype that is very similar to that observed in tkv mutants (comparewith panel G). I) Severe reduction in dpp function during wingdevelopment results in a failure of wing outgrowth. J) Ubiquitousmisexpression of the UAS-sog*5-2 mutant results in greatly reduced wingsize similar to that observed in the wings with greatly reduced Dppactivity (compare with panel I).

FIG. 3 depicts a molecular analysis of NOVA sog mutants. A) Diagram ofthe Sog protein. Sog is a large extracellular protein (1038 amino acids)consisting of four cysteine rich domains (CRs) defined by a conservedpattern of 10 cysteine residues. The first CR domain is located near theN-terminus of the protein and is separated by a long spacer from theremaining three CRs that are clustered at the C-terminus. B) Immunoblotanalysis of proteins extracted from flies carrying the full lengthUAS-sog construct or various mutant forms (UAS-sog*) under the controlof HS-GAL4. Protein extracts were prepared from flies with (+) orwithout (−) heat induction. Truncated forms of the Supersog protein areproduced from several of these mutants including the supersog-likcmutant sog*5-73 (FIG. 3F) and the more severe mutant sog*5-2, whichcauses a great reduction in wing size (FIG. 3J).

FIG. 4 demonstrates generation of dominant negative EGF-Receptormutants. Inventors employed a genetic scheme similar to that shown inFIG. 1A for mutagenizing a UAS-EGFR transgene using Δ2-3 transposase asthe mutagen in a small scale pilot screen and recovered two dominantnegative mutants, one of which is shown here (DN-EGFR2). AllUAS-transgenes were expressed ubiquitously using the MS-1096 GAL4 source(abbreviated G4 in this and subsequent figures). Inventors alsoconducted a small scale screen using EMS as the mutagen and recoveredone mutant (DN-EGFR3). In general, males (M) are more severely affectedthan females (F) due to dosage compensated expression of theX-chromosome linked MS-1096 GAL4 source. A) A wild type wing. B) A wingin which a wild-type UAS-EGFR transgene is expressed ubiquitously. Notethe presence of ectopic wing veins. C) A wing mis-expressing a UAS-rhotransgene. rho promotes high levels of EGF-R signaling and thereforegenerates a very strong ectopic vein phenotype in which most wing cellsadopt the vein fate. D) A female wing expressing the classic dominantnegative form of EGFR (UAS-DN-EGFR), which is an in vitro generatedconstruct that is truncated a short distance following the transmembrane(TM) domain (see middle row of panel P). In contrast to expression ofwild-type EGF-R, expression of DN-EGFR results in vein loss, which isparticularly acute for odd numbered veins, which form predominantly onthe dorsal surface of the wing where GAL4 expression is strongest. E) Afemale wing expressing the UAS-DN-EGFR2 mutant generated in the Δ2-3NOVA genetic screen. This mutant phenotype is very similar to thatresulting from expression of the classic DN-EGFR (compare to panel D).The precise nature of the lesion in the DN-EGFR2 mutant, which has beendetermined by PCR amplification of the mutated transgene and DNAsequencing (bottom row of panel P), results from a deletion of EGFRcoding sequences beginning at a site very near that used in creating theclassic DN-EGFR construct, which is then fused out-of-frame to part ofthe white gene. Consistent with this predicted protein structure forDN-EGFR2, a truncated EGFR band on immunoblots of fly protein extractsprepared from DN-EGFR2 expressing flies was observed. F) A female wingexpressing the UAS-DN-EGFR3 mutant generated in the EMS NOVA geneticscreen. This phenotype is noticeably weaker than that caused by theclassic DN-EGFR or DN-EGFR2 (compare with panels D and E). G) A malewing expressing the classic UAS-DN-EGFR mutant. This phenotype isstronger than that observed in females carrying a single copy of theX-linked MS 1096-GAL4 driver (compare to panel D). H) A female wingcarrying two copies of the X-linked MS 1096-GAL4 driver expressing theUAS-DN-EGFR2 mutant. This phenotype is approximately equal to that ofmales expressing a single copy of the GAL4 source, which issignificantly stronger than that of females carrying a single copy ofthe GAL4 driver (compare to panel E). I) A male wing expressing theUAS-DN-EGFR3 mutant. This phenotype is stronger than that observed infemales carrying a single copy of the X-linked MS 1096-GAL4 driver(compare to panel F). J) A wing co-expressing the classic DN-EGFR andwild-type EGFR constructs. DN-EGFR prevails in this situation (compareto panels B, E). K) A wing co-expressing the DN-EGFR2 and wild-type EGFRconstructs. EGFR largely prevails in this situation (compare to panelsB3) L) A wing co-expressing the DN-EGFR3 and wild-type EGFR constructs.A novel phenotype is observed in this situation, which consists of amuch more severe ectopic vein phenotype than observed with expression ofEGFR alone, and a significant reduction in wing size (compare to panelsB, F). The results presented in panels A-L suggest that DN-EGFR2 issimilar in activity to the classic DN-EGFR, albeit slightly weaker, andthat DN-EGFR3 is noticeably weaker than DN-EGFR and DN-EGFR2 and alsohas an activity that is distinct from the other two mutants (e.g. seepanel L). M) (SEQ ID NO:1) Diagram of wild-type UAS-EGFR construct (topline), UAS-DN-EGFR classic (middle line), and UAS-DN-EGFR2 (bottom line)depicting the locations of the p-element ends (arrow heads), the EGFRgene, and the white marker gene.

FIG. 5 demonstrates generation of loss-of-function revertants by EMSmutagenesis in a HS-rho transgene. A) EMS mutagenesis scheme to revertthe NOVA ectopic vein phenotype of rho30A flies, which results fromconstitutive misexpression of a wild-type rho transgene. B) A wing fromthe rho30A mutant, which has a moderate-strong ectopic vein phenotype.C) A wing from the rho30A* null revertant (class 0) in which the ectopicvein phenotype has been completely eliminated. D) A wing from a strong,but not null, rho30A* loss-of-function revertant (class 1), which hassome residual ectopic veins. E) A wing from a moderate, rho30A*loss-of-function revertant (class 3), which has significant residualectopic vein material. F) A diagrammatic map of Rho indicating thelocation and nature of all of rho30A* revertant mutations Inventors haverecovered and analyzed (SEQ ID NOS 2-6, 7-11, and 12-13, respectively inorder of appearance). Mutations are indicated by symbols above each rowof the figure as follows: class 0 null mutations (e.g. full reversion ofthe rho30A ectopic vein phenotype) are represented by an asterisk in row2, the first and third asterisks appearing in row 4, by the letter Dappearing above row 3, and by the letters L and R appearing above row 4;class 1 mutations with a small amount of residual NOVA rho activity arerepresented by the letters G, H, N, D and K appearing above row 2, theletter L appearing above row 3, the letter Y appearing above row 4, andthe second, fourth and fifth asterisks appearing above row 4; class 2mutations with a moderate degree of residual NOVA rho activity arerepresented by the letter W appearing above row 2, the letters S, A, G,I C and N appearing above row 3, and the letter D appearing above row 4;and class 3 mutations with significant residual activity, which isclearly less than that resulting from wild-type rho30A phenotype arerepresented by the letters I and T appearing above row 3, and the letterV appearing above row 4. These loss-of-function mutations tend to fallin TM domains and usually affect residues that have been highlyconserved in other Rho-related family members.

FIG. 6 shows how activated and dominant negative mutants are generatedin a UAS-rho transgene. Inventors employed a genetic scheme similar tothat shown in FIG. 1A for mutagenizing a UAS-rho transgene using Δ2-3transposase as the mutagen in a small scale pilot screen and recoveredseveral two NOVA mutants and a dominant negative mutant. A) A wild typewing. B) A wing mis-expressing a UAS-rho transgene, which generates avery strong ectopic vein phenotype. C) A wing from a rho[veinlet] viableloss-of-function mutant in which rho expression is nearly eliminatedduring larval development. Note that large sections of the distalportions of the L4 and L5 veins are missing as well as the tip of L3. D)A wing from a fly misexpressing the UAS-rho*6 mutant. In addition tobeing much smaller than a normal wing (panel A) or a wing expressingwild-type rho (panel B), many structures such as innervated bristles aremissing from the wing margin. E) A wing from a fly misexpressing theUAS-rho*7 mutant. This wing is much smaller than a typical wingexpressing the wild-type rho construct (compare with panel B) and mayencode an activated form of Rho. F) A wing from a fly misexpressing theUAS-rho*2 mutant. As this phenotype of this wing is very similar to thatof the loss-of-function rho[veinlet] mutant (compare with panel C), thismutant may encode a dominant negative form of Rho. G) Molecular analysisof the rho*6 NOVA rho mutant. The left panel shows DNA productsgenerated from amplification of wild-type versus rho*6 genomic DNAtemplates using convergent PCR primers located at the ends of the pUASconstruct. The band from rho*6 mutants is much smaller than thatamplified from wild-type. The right panel shows an immunoblot of proteinextracted from flies the expressing full length UAS-rho transgene or themutant UAS-rho transgene under the control of a heat induced HS-GAL4driver. As predicted from the PCR and DNA sequencing analysis (see panelH below), the Rho6 protein is highly truncated. H) Diagram comparing thestructures of the wild-type starting UAS-rho construct (top line) versusthat of the truncated UAS-rho*6 mutant (bottom line) (SEQ ID NO: 14).The conceptually translated Rho*6 mutant protein lacks the last six ofseven predicted TM domains and much of the loop between TM1 and TM2.This suggests that sequences near the N-terminus or in TM1 of Rho caninteract non-productively with some endogenous factor to generate theresulting NOVA phenotype.

FIG. 7 depicts how a NOVA screen uncovers novel activities of a UAS-rhotransgene. A) NOVA mutagenesis scheme. (B-G) Wings of the followinggenotypes: (B) wild type; (C) wing-GAL4/UAS-rhowt; (D)wing-GAL4/UASrho^(Neo′). Insets here and in F show interrupted margin(bracket) in stronger examples. (E) wing-GAL4AJAS-rho^(DN); (F)wing-GAL4/UAS-rho^(Neo)′; (G) rho^(ve). (H-K) Structures of wild-typeand mutant pUAS-rho constructs. Solid boxes indicate the transmembranedomains of the Rho protein. Triangles indicate the inverted terminalrepeats of the P element. (H) Wild-type pUAS-rho^(wt); (I)pUAS-rho^(Neo)(SEQ ID NO: 14); (J) pUAS-rho ^(DN); (K) immunoblotanalysis of protein extracts from wild-type (lane 1), pHS-rho (lane 2),pHS-GAL4/UAS-rho^(Neo) (lane 3), and pHS-GAL4/UASrho^(DN) (lane 4) adultflies submitted to a 1-h heat shock at 38° C. (L) Northern blot of mRNAextracted from wild-type (lane 1), pHS-GAL 4/UAS-rhowt (lane 2), andpHS-GAL4/UAS-rho^(DN) (lane 3) adult flies following a 1-h heat shock.

FIG. 8 shows how rho^(Neo) interferes with Notch signaling. (A-D) Wingsof the following genotypes: (A) vg-GAL4/UAS-rho^(Neo); (B) N^(55e11)/+;(C) N^(Axl)/y (male); (D) N^(Axl)/+ wing-GAL4/UAS-rho^(Neo) (female);(E) wingless expression along the wing margin (M) of a wild-type thirdlarval instar imaginal disk; (F) absence of wg expression inwing-GAL4/UAS-rho^(Neo) discs (arrow, margin primordium); (G) Cutexpression along the wing margin (M) of a third instar imaginal disk;(H) reduced Cut expression in a wing-GAL4/UAS-rho^(Neo) wing disk(arrow, margin primordium).

FIG. 9 demonstrates that rho^(DN) functions by an RNAi-like mechanism.(A) MAPK activation in a wild-type third instar imaginal disk inlongitudinal vein primordia (L2-L5) and wing margin (M). (B) MAPKactivation in a wing-GAL4>UAS-rho^(wt) disk. (C) Lack of MAPK activationin a wing-GAL4>UAS-rho^(DN) disk. (D) rho RNA expression in awing-GALteUAS-rho^(wt) disk. (E) Undetectable rho RNA expression in awing-GAL4>UAS-rho^(DN) disk. Endogenous rho expression (Inset) is alsolost. (F) rho expression in a wing-GAL4>UAS-rho^(wt)/UAS-rho^(DN) disk.(G) Rho protein expression in a GAL4>UAS-rho wing disk. (H) Strongreduction of Rho expression in a wing-GAL4_UAS-rho/UAS-rho^(DN) disk.(I) wing-GAL4>UAS-rho wing. (J) wing-GAL4>UAS-rho^(wt)/UAS-rho^(DN)wing.

FIG. 10 depicts generation of dominant-negative and ^(Neo)morphic Starmutants. (A-H) Wings of the following male genotypes: (A)wing-GAL4>UAS-Star, (B) wing-GAL4>UAS-Star^(DN)/UAS-Star^(DN); (C)wing-GAL4>UAS-Star^(DN)/+; (D) wing-GAL4>UAS-Star^(DN)/UAS-Starwt; (E)wing-GAL4>UAS-Star^(DN)/UAS-rhowt; (F)wing-GAL4>UAS-Star^(DN)/UAS-m-grk(2×); (G) wing-GAL 4>UAS-Star^(Neo1);(H) wing-GAL4(2×)>UAS-Star^(Neo2)/UAS-Star^(Neo2). (I-K) Structure ofwild-type and mutant pUAS-Starwt constructs. (I) pUAS-Starwt, the bluebox indicates the single transmembrane domain of Star. (J)pUAS-Star^(DN) (SEQ ID NO: 15) a 1.9-kb inversion with breakpoints inboth the Star and 3′ untranslated simian virus 40 sequences of pUAStresults in a C-terminal truncation of the Star protein. (K)pUAS-Star^(Neo1) (SEQ ID NO: 16) and pUAS-Star ^(Neo2)(SEQ ID NO: 17).

FIG. 11 shows the generation of dominant-negative forms of UAS-Egf-r.(A-F) Wings of the following male genotypes: (A)wing-GAL4>UAS-Egf-r^(wt); (B) wing-GAL4/>E-r^(DN1); (C)wing-GAL4>Egf-r^(DN2); (D) wing-GAL4>Egf-r^(DN3), (E)wing-GAL4>Egf-r^(DN2)/Egf-rwt; (F) wing-GAL 4>Egf-r^(DN3)/Egf-r^(wt).(G-J) structures of wild-type and mutant pUAS-Egf-r constructs: (G)pUAS-Egf-r^(wt) (H) pUAS-Egfr^(DN1), which has a STOP codon 20 aminoacid after the transmembrane domain; (I) pUAS-Egf-r^(DN2), which has abreakpoint mapping 13 amino acid downstream of that of Egf-r^(DN2)(SEQID NO: 1); (J) pUAS-Egf-r^(DN3), which has a point mutation (R1062K) inthe kinase domain (SEQ ID NO: 18). Stars (*) indicate consensus residuesconserved across a wide variety of tyrosine kinases.

FIG. 12 shows that many bacterial toxins modify Rho-like GTPases atspecific residues, which are conserved in Rho/Rac fly homologues (SEQ IDNOS: 19-27, respectively in order of appearance).

FIG. 13 is a basic scheme for creating Dominant-Negative alleles ofbacterial toxins.

FIG. 14 is a variant genetic scheme using chemical mutagenesis

DESCRIPTION OF THE PREFERRED EMBODIMENTS Introduction

Abbreviations: EGF-R, epidermal growth factor receptor; MAPK,mitogen-activated protein kinase; NOVA, novel overexpression activity;RNAi, RNA interference; UAS, upstream activation sequence; EMS, ethylmethanesulfonate.

The instant invention is a novel genetic method for generating a noveloverexpression activity (hereinafter “NOVA”) allele of a gene ofinterest (gene-x). Novel overexpression activity (NOVA) alleles of agene of unknown function are powerful genetic tools that can provideimportant insights into the function of that gene. In addition, once aNOVA allele of a gene has been isolated, it is typically possible todesign mutant screens to identify second site mutations in other genesacting in the same pathway as the gene of interest. Mutants in thesesecond site modifier loci alter the NOVA phenotype, either by enhancingor suppressing that phenotype. Finally, it is possible to use mutagensto revert NOVA mutant phenotypes generated by this method and therebyefficiently generate second-site intragenic loss-of-function alleleswithin that gene of interest for structure/function studies.

The first step in this genetic method is to misexpress a wild-type (e.g.normal) copy of a gene in Drosophila and determine the phenotypicconsequence of doing so. This type of analysis is routine using eitherheat inducible expression system (HS-gene-x) or the GAL4/UAS expressionsystem (UAS-gene-x) of Brand and Perrimon (Brand and Perrimon, 1993).Once the phenotype due to misexpressing gene-x has been determined,which may not differ from wild-type, the novel idea is to:

1) Mutagenize flies carrying the gene-x transgene using chemicalmutagens, radiation, or transposase induced mutagenesis.

2) Activate global or restricted expression (e.g. in time or space) ofthe mutagenized transgene (e.g. by heat inducing flies carrying aHS-gene-x transgene or by crossing flies carrying a UAS-gene-x transgeneto one of the many available GAL4 drivers).

3) Screen for new dominant phenotypes resulting from expression of themutagenized transgene-x such as constitutively active or dominantnegative forms of the gene. Two examples of the use of this method aresummarized below.

An important difference between this earlier screen and the NOVAmutagenesis described here is the use of a high-level expression system,which Inventors believe is critical for the recovery of novel activitiesthat would otherwise go undetected.

The advantage of this method over existing methods is the efficiency andspeed with which it can be applied to a wide variety of genes. Moreover,because our method is not predicated on assumptions based on proteindomain structure and function, we can recover mutants in an unbiasedfashion.

The Drosophila epidermal growth factor receptor (EGF-R) controls manycritical cell-fate choices throughout development. Several proteinscollaborate to promote localized EGF-R activation, such as Star andRhomboid (Rho), which act sequentially to ensure the maturation andprocessing of inactive membrane-bound EGF ligands. To gain insights intothe mechanisms underlying Rho and Star function, a mutagenesis schemewas developed to isolate novel overexpression activity (NOVA) alleles.In the case of rho, a dominant ^(Neo)morphic allele was isolated, whichinterferes with Notch signaling, as well as a dominant-negative allele,which produces RNA interference-like flip-back transcripts that reduceendogenous rho expression. Also obtained were dominant-negative and^(Neo)morphic Star mutations, which have phenotypes similar to those ofrho NOVA alleles, as well as dominant-negative Egf-r alleles.

Materials and Methods

Fly Stocks and Crosses.

Upstream activation sequence (UAS)-rho and pUAS-Star stocks weredescribed in Guichard, A. et al (1999), the contents of which areincorporated herein. The pUAS-Egf-r and pUAS-Egf-r^(DN1) stocks wereprovided by Allan Michelson (Brigham and Women's Hospital, Boston). Allcrosses were performed at 25° C.

Molecular Analysis of Mutations in rho, Star and Egf-r Transgenes.

For analysis of Δ2-3-induced mutants, sets of primers corresponding tosequences in the pUASt vector, the rho cDNA, or the Star cDNA were usedto search for alterations in the various mutants by standard PCR orinverse PCR, with the Long Expand PCR system (Roche MolecularBiochemicals catalogue no. 1681842).

Immunoblot Analysis of Mutant Rho Proteins.

The anti-Rho serum (Sturtevant et al (1996)) was used for immunoblottingat 1/1,000 dilution, in 0.25% Tween 20, 1% milk in PBS. Secondaryantibodies (horseradish peroxidase-coupled anti-rabbit IgG, JacksonImmunoResearch catalogue no. 111-035-003) were used at 1/5,000 dilution.Chemiluminescent detection was performed by using the Supersignal kit(Pierce catalogue no. 34080).

In Situ Hybridization and Histochemistry.

In situ hybridization, histochemistry (O'Neill, J. & Bier, E. (1994)),and detection of MAPK activation (Guichard, A. et al (1999)), bothreferences incorporated herein, were performed as described therein.Anti-Cut antibodies were obtained from the Developmental StudiesHybridoma Bank (University of Iowa).

Northern Analysis

Northern blots were prepared by using standard methods, hybridized witha horseradish peroxidase-labeled rho RNA probe, and detected by usingthe Chemiluminescent system CDP-Star (Amersham Pharmacia catalogue no.RPN3690).

General Genetic NOVA Screening Method

Male flies carrying a transgene-x are mutagenized, mated en masse torelevant female strains (see below for examples) and the progeny arethen screened for novel NOVA phenotypes (e.g. phenotypes differing fromthat caused by misexpressing the wild-type transgene-x). The mutagenemployed can be of any suitable type, including, but not limited to,chemical, X-ray

EXAMPLE 1

Mutagen=ethyl methane Sulfonate (EMS)

Transgene=HS-gene-x or p UAS-gene-x

i. white-males carrying a white+ marked transgene-x on an autosomalchromosome denoted

w−; p[w+gene-x]/p[w+gene-x] are crossed en masse to white-females (forHS-gene-x constructs) or white-; GAL4/GAL4 females (for UAS-gene-xconstructs) at a ratio 25 mutagenized males to 50 females in bottles offly food.

ii. Single progeny of this cross carrying a mutagenized transgene andthe linked balancer chromosome denoted w−; p[w+gene-x*]; ÷GAL4 are thenscreened for a NOVA phenotype.

iii. Candidate NOVA mutants are then crossed to the appropriate balancerfemales to establish stable stocks, which are then retested fordependence on heat induction of GAL4 and analyzed further.

See FIG. 4 for an example of EMS induced mutations generated in aUAS-EGFR transgene to generate a dominant negative form of theEGF-Receptor. This method was also used (FIG. 5A) to generateloss-of-function rho alleles by mutagenizing flies carrying aconstitutively active HS-rho construct that have ectopic veins as aconsequence of the overexpression of the rho transgene (FIG. 5B) withEMS. Screening for revertants that have loss-of-function mutations inthe rho transgene was performed (FIG. 5C-E). These rho revertant mutantshave been sequenced to determine what regions of the Rho protein arecritical for its ability to promote EGF-R signaling (FIG. 5F).

Advantage of EMS as a Mutagen

It is a relatively random mutagen that typically causes single aminoacid substitutions.

Disadvantage of EMS as a Mutagen

It has no intrinsic bias for targeting the transgene versus all otherendogenous genes for mutagenesis.

EXAMPLE 2

Mutagen=p-element Transposase enzyme

Transgene=pUAS-gene-x

i. w−; p[w+UAS-gene-x]/p[w+UAS-gene-x] flies are crossed to fliescarrying an activated form of transposase denoted w−; A2-3 Sb/TM6.

ii. Males of the genotype: w−; p[w+UAS-gene-x]; A2-3 Sb (transgene-x onthe X-chromosome, second chromosome, or fourth chromosome) or w−;p[w+UAS-gene-x]/Δ2-35b (transgene-x on the third chromosome) are crossedto w−; GAL4/GAL4 females (the GAL4 driver of choice can be located onany chromosome) at a ratio of 3 mutagenized males to 8 females in vialsof fly food (vials are used instead of bottles to insure thatindependent Δ2-3 induced mutations are recovered separately).

iii. Candidate NOVA mutants of the genotype w−; p[w+UAS-gene-x]; GAL4 or

w−; p[w+UAS-gene-x]/GAL4 are then crossed to the appropriate balancerfemales to establish stable stocks, which are then retested for GAL4dependence and analyzed further.

Examples are shown of dominant NOVA and dominant negative phenotypesobtained from subjecting the UAS-sog (FIG. 2), UAS-EGFR (FIG. 4) andUAS-rho (FIG. 6) transgenes to Δ2-3 mutagenesis.

Advantage of Transposase as a Mutagen

Transposase induces a high frequency of mutations in transposablep-elements and thus in p-element based vectors containing transgenes(Daniels et al., 1985). This is a very efficient method for directingmutations to the transgene of interest.

Disadvantage of Transposase as a Mutagen

Transposase most frequently causes deletions and other large scalechromosomal aberrations rather than more restricted mutations such asamino acid substitutions.

Generation of Novel Mutations in Human Genes

The NOVA method can be used to generate novel mutations in human genesthat can provide insight into the function of these genes and lead tothe identification of medically relevant partners of these genes. Thismethod is generally applicable because it can result in the efficientgeneration of activated and/or dominant negative NOVA-type mutations ina broad variety of genes. Since nearly all proteins interact with atleast one other protein or substrate, it should be possible, inprinciple, to generate dominant negative alleles of virtually every geneif the mutant protein product interacts non-productively with suchpartners and is expressed at sufficiently high levels in the right placeand time to titrate out those partners. Mutated transgenes can besequenced to identify altered amino acid residues that are involved inpotential protein-protein interactions. This information could then beused to design wild-type versus mutant peptides for yeast two hybridscreens to identify the putative protein partners that interact with theprotein of interest. In contrast to this novel NOVA method, previouslyexisting methods for generating dominant mutant phenotypes in modelorganisms such as Drosophila to study the function of a gene of interestare typically limited to the smaller subset of genes for whichmisexpression of the wild-type version of that gene generates aphenotype.

The NOVA method is expected to be applicable to other organismsincluding humans and model systems such as yeast, C. elegans, andArabidopsis. Regarding potential applications to human disease, itshould be possible to misexpress any of the over 1,000 currentlyidentified human disease genes in Drosophila, and to use the NOVA methodto isolate novel NOVA alleles of these genes. Once such a NOVA allele ofa human disease gene has been isolated, it would be possible to performsecond-site modifier screens to search for mutations in other genes,that when heterozygous, either enhance or suppress the human diseasegene NOVA phenotype. This classic and effective use of Drosophila as amodel genetic system for identifying all genes acting in a given pathwayshould reveal the identity of many Drosophila genes which interact withhuman disease genes. The majority of these Drosophila genes are expectedto have human homologs that function equivalently, since over 50% ofhuman disease genes have clear homologs in Drosophila (Reiter et al.,2000). Because the function of many of human disease genes and thepartners with which they interact are typically unknown, Inventorsanticipate that this NOVA genetic method will be a powerful tool foridentifying and dissecting genetic pathways involved in human disease.

With respect to plants, isolation of NOVA alleles of plant genescontrolling agriculturally relevant traits could lead to the developmentof new commercially valuable strains of plants. These NOVA alleles wouldalso be valuable tools for conducting second-site modifier screens inArabidopsis, analogous to those outlined above for Drosophila, toidentify additional genes involved in controlling agriculturallydesirable traits.

EXAMPLE 3

Isolation of rho Overexpression Alleles.

Rho has been recently defined as a novel type of intramembrane serineprotease that cleaves the membrane-bound ligand Spi (mSpi) in the Golgiapparatus before its release into the extracellular space. Although acatalytic domain and key residues essential for proteolytic activityhave been defined in Rho (Urban et al (2001)), it is not known whichother parts of the protein fulfill regulatory functions and interactwith functional partners such as Star or other components. Like manyproteins recently identified in the Drosophila genome project, Rho doesnot contain any signature domains that could give clues about possibleprotein-protein interactions. In this context, Inventors developed a newstrategy to screen for potential NOVA alleles of rho, which mightprovide additional insights into the mode of Rho action. The NOVA methodmakes use of the two-component GAL4/UAS expression system (Brand, A. H.& Perrimon, N. (1993)). The principle of this scheme is to expose a UAStransgene of interest to mutagenesis, express the mutated transgene inthe F₁ progeny at high levels in a desired pattern by using a strongGAL4 driver, and then screen for novel visible phenotypes. In thepresent case, Inventors exposed a UAS-rho transgene to the Δ2-3transposase, which induces rearrangements such as small deletions,inversions, and duplications within or adjacent to P element insertionsas a byproduct of gap repair after excision events (Daniels et al(1985); Delattre et al (1995)). Inventors crossed individuals carryingthe potentially mutagenized UAS-rho*transgene to flies carrying a strongubiquitous wing-specific GAL4 driver (MS1096GAL4, referred to aswing-GAL4 hereafter) and then screened for novel dominant phenotypes inapproximately 15,000 F1 progeny of this second cross (FIG. 7A). Amongthe individuals of the relevant wing-GAL4/UAS-rho* genotype, most fliesexhibited the all-vein phenotype resulting from strong misexpression ofunaltered wild-type UAS-rho (FIG. 7C). A smaller fraction of the F1progeny had a wild-type phenotype, likely to reflect precise pUAS-rhoexcision events (FIG. 7B). In addition, Inventors recovered twoindividuals exhibiting distinct dominant NOVA phenotypes. The firstmutant has small blistered wings with thickened veins and margin defects(FIG. 7D Inset). In the most-affected individuals, wings are virtuallyabsent. Because loss-of-margin structures and great reduction in wingsize are not phenotypes observed in loss-of-function rho mutants or inflies misexpressing wild-type rho, Inventors refer to this ^(Neo)morphicrho mutant as rho^(Neo). The second rho mutant had missing distalportions of wing veins (FIG. 7E) typical of rho loss-of-functionsituations (e.g., rhove, FIG. 7G). Inventors considered this NOVA mutantto be a likely dominant-negative form of rho (rho^(DN)).

EXAMPLE 4

Characterization of the Molecular Lesions in rhoNOVA Mutants.

As a first step in analyzing the new rho NOVA alleles, Inventorsconfirmed that the rho^(Neo) and rho^(DN) phenotypes wereGAL4-dependent. Inventors then used combinations of PCR primer sets toamplify rho sequences within the pUAS vector and/or surrounding genomicsequences to identify molecular lesions responsible for the dominantrho^(Neo) and rho^(DN) activities. This analysis revealed that theUAS-rho^(Neo) mutant carries a 5-kb deletion removing parts of both therho cDNA and the adjacent white marker gene. This rho^(Neo) mutantconstruct is predicted to encode a truncated protein containing thefirst 140 amino acids of Rho (including the N terminus, TM1, half of thefirst loop) and 10 amino acids encoded out-of-frame by the 3′ end of thewhite gene, which are fused to the rho coding sequence (FIG. 7I, comparewith wildtype structure in FIG. 7H).

Consistent with the predicted structure of Rho^(Neo), immunoblotting ofprotein extracts from heat-induced HSGAL4>rho^(Neo) flies by using anN-terminal specific anti-Rho antibody (Sturtevant et al (1996)) revealedhigh levels of a shorter-than-normal Rho protein (27 kDa instead of 43kDa for the wild-type species; FIG. 7K, lanes 3 and 2, respectively).Interestingly, this Rho^(Neo) protein comigrates with a smaller Rhoprotein species that is consistently observed on heat induction of fulllength Rho expression (FIG. 7K, lane 2).

Misexpression of components in the EGF-R pathway using the wing-GAL4driver does not typically result in margin defects. It was thereforeimportant to verify that the observed rho^(Neo) phenotype resulted frommisexpression of the truncated rho mutant transgene rather than fromsome adjacent genomic sequence. Although the pUASt vector does notactivate expression of endogenous genes efficiently (Rørth et al(1998)), a general potential caveat to the NOVA method is thatGAL4-dependent phenotypes could occasionally result from misexpressionof an unrelated gene near the chromosomal site of a pUAS insertion. Toaddress this concern, Inventors cloned a PCR product containing thetruncated cDNA of the rho^(Neo) gene back into the pUASt vector andretransformed this construct (named UAS-rho^(Neo′)) into flies.Misexpression of UAS-rho^(Neo′) with the wing-GAL4 driver resulted inthe same phenotype (FIG. 7F) as that obtained with the initialUAS-rho^(Neo) isolate (FIG. 7D). Inventors conclude that the truncatedrho^(Neo) allele is indeed responsible for the observed wing phenotypes.

Molecular analysis of the UAS-rho^(DN) mutation revealed an invertedduplication of the UAS and rho sequences with a spacer portionconsisting of rho sequences (FIG. 7J). This UAS-rho^(DN) mutant ispredicted to generate an RNA with a hairpin structure (Guichard et al.PNAS), which potentially could exert an RNA interference (RNAi) effect(see below). Consistent with this prediction, a larger rho transcript(≈3.9 kb) observed in flies expressing rho^(DN) (FIG. 7L, lane 3) thanin those expressing wild-type rho (2.3 kb; FIG. 7L, lane 2).

EXAMPLE 5

rho^(Neo) Interacts with the Notch Pathway.

Ubiquitous expression of rho^(Neo) in the wing primordium disruptsformation of margin structures, suggesting that it interacts with apathway involved in inducing margin cell fates or differentiation. Toidentify phenotypes specifically attributable to defects in wing margincell fates, UAS-rho^(Neo) with the margin-specific vestigial-GAL4 driverwas expressed and a more pronounced loss of margin structures wasobserved (FIG. 8A) esembling that associated with reduction of wingless(wg) or Notch function (FIG. 8B). In addition, ubiquitous misexpressionof UAS-rho^(Neo) results in thickened veins (FIG. 7D)—another signaturephenotype of Notch pathway mutants (FIG. 8B). Although ectopic orthickened veins can also result from misexpression of full-length rho(22,29), it is unlikely that rho^(Neo) function is mediated byactivation of EGF-R signaling or deregulated Rho protease activity,because this mutant lacks all sequences necessary for the proteolyticfunction of Rho (Lee et al (2001); Urban et al (2001)). It has also beenreported that a dominant-negative form of EGF-R misexpressed in themargin causes notching (Nagaraj et al (1999)). However, becauseloss-of-function rho⁻ or Egf-r⁻ clones do not result in marginphenotypes (Diaz-Benjumea & Garcia-Bellido (1990); Diaz-Benjumea & Hafen(1994)), and because such loss-of-function mutations do not generateectopic veins as observed in wing-GAL4/rho^(Neo) wings, it seemsunlikely that rho^(DN) functions by a dominant-negative mechanismspecific to the EGF-R pathway. The hypothesis that rho^(Neo) interfereswith Notch signaling was tested by assaying the expression of twodownstream targets of Notch, wg and cut. In wild-type discs, wg and Cutare expressed in a narrow row of margin cells at the dorso-ventralboundary (FIGS. 8 E and G; Couso et al (1994) and Micchelli et al(1997)). In wing discs ubiquitously expressing the UAS-rho^(Neo)construct, expression of both wg and Cut was abolished or significantlyreduced (FIGS. 8 F and H), suggesting that rho^(Neo) reduces Notchsignaling. Consistent with this proposal, all rho^(Neo) phenotypes arestrongly suppressed by one copy of the activated N^(Axl) allele inheterozygous females (FIG. 8D, compare with FIG. 7D). This N^(Axl)allele, on its own, causes loss of veins when homozygous or hemizygous(FIG. 8C; Lindsley, D. & Zimm, G. (1992)), but has little effect whenheterozygous.

One explanation for the rho^(Neo) mutant phenotype is that it interfereswith other Rho-related proteins (Wasserman et al (2000)). Consistentwith this possibility, a human Rhomboid-like protein has been reportedto bind the Notch-activating protease Presenilin (Pellegrini et al(2001)), suggesting a further connection between Rho-related proteinsand Notch signaling. To determine whether Rho^(Neo) interferes directlywith the Notch pathway (e.g., by binding to a component of the Notchpathway and blocking its activity), or indirectly by impinging on otherpathways interacting with Notch to promote margin development and veinformation, additional analysis will be required.

An important question regarding the biological significance of Rho^(Neo)is whether a similar Rho fragment is generated in vivo and mediates acomponent of endogenous rho activity. It is relevant in this regard thatan N-terminal Rho fragment of nearly identical size to rho^(Neo) isconsistently produced in vivo when full length Rho is overexpressed(FIG. 7K) and is also present in wild-type embryo extracts. Onepotential biological role for this N-terminal fragment of Rho could bethe suppression of Notch activity in cells expressing high levels ofRho. Such negative feedback might aid in the creation of mutuallyexclusive domains of Notch and EGF-R activity in some developmentalsettings, as has been recently suggested for the partition of the eyedisk into antenna and eye fields (Kumar & Moses (2001)) and for bristledifferentiation (Culý' et al (2001)).

EXAMPLE 6

rho^(DN) Inhibits EGF-R Activity by an RNAi-Like Mechanism.

To confirm that rho^(DN) acts via a dominant-negative mechanism byinhibiting endogenous rho expression, and hence EGF-R activity, in situactivation of MAPK in wing discs expressing UAS-rho^(DN) was examined.In the wing disk, MAPK activation revealed by an antibody specific forthe diphosphorylated MAPK (Gabay et al (1997)), is restricted to veinand margin primordia and depends on localized rho expression andsubsequent activation of EGF-R signaling (FIG. 9A; Guichard et al(1999); Gabay et al (1997) and Martý'n-Blanco et al (1999)). In linewith rho expression defining the domain of EGF-R/MAPK signaling,ubiquitous activation of MAPK is observed in response to misexpressionof UAS-rho (FIG. 9B; Guichard, A. et al (1999)). In contrast,overexpression of UAS-rho^(DN) abolishes MAPK activation in, both, veinand margin primordia (FIG. 9C), consistent with rho^(DN) inhibitingendogenous rho expression and subsequent EGF-R activation.

In support of rho^(DN) functioning by an RNAi-like mechanism (Carthew(2001)), Rho protein could not be detected in extracts from theheat-induced UAS-rho^(DN) flies (FIG. 7K, lane 4). In addition,UAS-rho^(DN) transgene-derived RNA expression was nearly undetectable byin situ hybridization in wing-GAL4/UAS-rho^(DN) wing imaginal discs(FIG. 9E), consistent with the formation of double-stranded RNA hairpinstructures that are then degraded to 21- to 23-nt fragments, which wouldbe inaccessible to hybridization. Critically, endogenous rho expressionin vein and margin primordia (FIG. 9E Inset) was also absent in thesediscs, indicating that rho^(DN) interferes with the expression orstability of the endogenous rho mRNA. A wild-type UAS-rho transgene wasalso coexpressed with UAS-rho^(DN) and a significant overall reductionin the level of the wild-type rho mRNA (FIG. F, compare with D) and onlya faint trace of Rho protein staining (FIG. 9H) relative to thatproduced by the wild-type UAS-rho transgene alone (FIG. 9G) wasobserved. The fact that Rho protein levels were more severely reducedthan rho RNA levels in discs coexpressing rho and rho^(DN) suggests thatrho^(DN) compromises translation of the wild-type rho RNA as well asreducing its stability. In line with these various observations, theall-vein phenotype of wings misexpressing wild-type UAS-rho (FIG. 9I)appeared almost completely suppressed by coexpression with UAS-rho^(DN)(FIG. 9J). Cumulatively, these data indicate that rho^(DN) acts byinhibiting the activity of rho, most likely by promoting the degradationof its RNA and blocking its translation. This example illustrates thepotential utility of NOVA mutagenesis for creating an RNAi version of agene of interest by purely genetic means, as an alternative to the timeconsuming and often problematic construction of RNAi inserts by in vitroengineering.

EXAMPLE 7

Isolation of NOVA Alleles of Star.

Because novel dominant rho alleles were readily generated by NOVAmutagenesis, Inventors wondered whether this scheme could besuccessfully applied to other UAS transgenes of interest. Because Starcollaborates with rho and has no signature domain indicative of itsfunction, it too is a good candidate for NOVA mutagenesis.Overexpression of wild-type Star typically results in no phenotype inthe wing other than faint ectopic vein material near longitudinal veins(FIG. 10A). A UAS-Star transgene was submitted to the Δ2-3 transposaseand the same wing-GAL4 driver was used to misexpress mutagenized Starinsertions at high levels. In this screen of ≈15,000 progeny, Inventorsrecovered two types of GAL4-dependent dominant mutant alleles, referredto as Star^(DN) and Star^(Neo), which resulted in phenotypes nearlyidentical to those produced by misexpression of rho^(DN) and rho^(Neo).Thus, ubiquitous misexpression of Star^(DN) results in vein truncation(FIGS. 10 B and C) similar to that observed in wings lacking Staractivity (Guichard, A. et al (1999)), whereas misexpression ofStar^(Neo) alleles causes reduction in wing size, thickened veins,blisters, and strong disruption of margin structures (FIGS. 10 G and H).Consistent with Star^(DN) acting in a dominant-negative fashion,coexpression of this mutant with wild-type Star suppresses the vein-lossphenotype of Star^(DN) (FIG. 10D, compare with C). The nearly identicalphenotypes produced by NOVA alleles of the rho and Star transgenesprovide further evidence for the intimate functional relationshipbetween rho and Star (Guichard, A. et al (1999)).

Molecular analysis of one Star^(DN) mutation revealed that it isassociated with an inversion, which has breakpoints mapping within theStar coding sequence and its 3′ untranslated regions. As a result, theStar^(DN) transgene is predicted to encode a shortened protein with alarge C-terminal truncation preceding the single transmembrane domain ofStar at amino acid 124 (FIG. 10J, compare with wild-type structure inI). Star is a predicted type II membrane protein with a cytoplasmic Nterminus, suggesting that the Star^(DN) peptide would be free in thecytoplasm. The C terminus of Star is required for binding to mSpi(Martý'n-Blanco et al (1999)) and promoting its transport from theendoplasmic reticulum to the Golgi (Lee et al (2001)). Because Star^(DN)lacks the sequences necessary for interaction with mSpi, it is unlikelythat it acts by titrating out Spitz-like ligands in an unproductiveinteraction. Consistent with this expectation, coexpression of Star^(DN)with mSpi or mGrk did not restore a wild-type phenotype but ratherresulted in increased vein-loss (FIG. 10F). It seems more likely,therefore, that the N-terminal region of Star normally interacts withanother partner required for Spi maturation (e.g., Rho), and that theStar^(DN) fragment binds and sequesters this partner in an inactivecomplex. In support of this view, coexpression of Star^(DN) with rhoefficiently suppresses rho-induced ectopic veins (FIG. 10E). Star maythus function in a multimeric complex that includes mSpi and Rho and/orsome other component. According to this model, coexpression of Star^(DN)with mSpi or mgrk would result in a greater titration of this endogenousfactor and in an enhanced phenotype, as observed in FIG. 10F.

Inventors also analyzed the structures of two Star^(Neo) mutants andfound lesions mapping virtually to the same location. In both cases, adeletion fused C-terminal sequences of Star with the white marker. As aconsequence, Star^(Neo1) and Star^(Neo2) encode Star proteins lacking 15and 17 C-terminal amino acids, respectively, followed by differentresidues encoded by frameshift fusions to distinct white sequences (FIG.10K). As in the case of the rho^(Neo) mutant, overexpression ofStar^(Neo1) resulted in loss of wg and Cut expression in the marginprimordia of third instar larval discs (data not shown). ThatStar^(Neo2) is somewhat weaker than Star^(Neo1) in inducing Notch-likephenotypes may result from the different C-terminal amino acids encodedby white sequences or may reflect a function of the two amino acidspresent in Star^(Neo1) that are missing in Star^(Neo2).

The strong similarity between rho^(Neo) and Star^(Neo) phenotypessuggests that they arise from interference with a common process. As arecent report shows that Star and Rho act sequentially rather thansimultaneously (Lee et al (2001)), it is possible that the component(s)interacting with rho^(Neo) and Star^(Neo) act in the Golgi apparatus,where Star may hand over mSpi or other substrates to Rho for the finalstep of maturation.

EXAMPLE 8

Recovery of Distinct Classes of Dominant-Negative Egf-r Alleles in NOVAScreens.

The Egf-r gene is another good test case for NOVA analysis, because invivo generated NOVA alleles could be compared with an existingdominant-negative Egf-r (Egf-r^(DN1)) construct created by in vitrogenetic engineering (42). Strong ubiquitous expression of Egf-rwt in thewing results in moderate ectopic vein formation (FIG. 11A), whereasoverexpression of Egf-r^(DN1) results in small wings and vein loss (FIG.11B). EGF-R^(DN1) is a truncated version of the EGF-R lacking thecytoplasmic kinase domain (FIG. 11H, compare with Egf-rwt in G) and hasbeen proposed to act primarily by forming nonfunctional heterodimerswith endogenous wild-type EGF-R chains (Kashles et al (1991)). Inventorssubmitted the UAS-Egf-rwt transgene (FIG. 11G) to mutagenesis usingeither Δ2-3 transposase or ethyl methanesulfonate (EMS), and crossed themutagenized males to wing-GAL4 females in two screens of 4,000individuals each. New putative dominant-negative Egf-r alleles wererecovered in both screens, which Inventors refer to as Egf r^(DN2) andEgf-r^(DN3), respectively. Misexpression of either of the pUAS-Eg-r^(DN)alleles with the wing-GAL4 driver results in small curved wings and veintruncations (FIGS. 11 C and D), which are similar to, although somewhatweaker than, the phenotypes generated by misexpression of the referenceEgfr^(DN1) construct (FIG. 11B).

Molecular analysis of the Δ2-3 induced Egf-r^(DN1) showed that thismutant is deleted for the entire kinase domain of the receptor and asmall portion of the white marker, resulting in an “out-of-frame” fusionbetween the two genes, mapping 33 amino acids after the transmembranedomain of EGF-R (FIG. 11I). This structure is very similar to that ofEgf-r^(DN1), which has a STOP codon 20 amino acids after thetransmembrane domain (FIG. 11H). The EMS-derived Egf-r^(DN3) mutationresults from a single amino acid substitution, R1062K, in the kinasedomain of the receptor (FIG. 11J), consistent with the propensity of EMSto act as a point mutagen. This substitution is conservative; however,it alters a residue that is absolutely invariant among all tyrosinekinases and is immediately adjacent to the catalytic aspartate residuein the active site of the kinase domain (Johnson et al (1998)). AlthoughEgf-r^(DN2) and Egf-r^(DN3) generate similar phenotypes in the wing, itwas found that their activities differ when expressed with Egf-rwtCoexpression of Egf-r^(DN2) (or Egf-r^(DN1)) with Egf-rwt results inmutual suppression of the Egf-rwt ectopic vein phenotype and of theEgf-r^(DN2) vein-loss phenotype (FIG. 11E). In contrast, Egf-r^(DN3) didnot suppress the Egf-rwt phenotype, but instead interacted positively toproduce smaller rounded wings with a strong all-vein phenotype (FIG.11F). As the Egf-r^(DN3) mutant retains a complete cytoplasmic domainthat could interact with effector molecules, coexpression of this mutantwith Egf-rwt may lead to the formation of EGF-R^(DN3)-EGF-R^(wt)heterodimers with partial activity, as has been suggested for Egf-ralleles that exhibit interallelic complementation (Raz et al (1991)).

Perspectives on the General Utility of the NOVA Method.

An attractive feature of the NOVA mutagenesis scheme described above isthat the transgene of interest is modified in vivo, without the need ofin vitro genetic engineering followed by time-consuming transformationprocedures. More critically, NOVA mutants are recovered in an unbiasedfashion solely based on the nature of the phenotypes they induce. Thisphenotype-based screening eliminates the need for making predictionsregarding likely functions of specific protein domains. Given that asubstantial fraction of proteins predicted by genome sequencing haveyet-unknown functions, the ability to screen for mutants without anyadvance knowledge of functional domains should be of significantutility. Although transposase is a particularly efficient mutagen forNOVA screening because it targets P element sequences, EMS can also beused in this scheme to create random point mutations. The differentbehaviors of the truncated Egf-r^(DN2) and substitution Egf-r^(DN3)mutants, when coexpressed with wild-type Egf-r, illustrate the value ofscreening for mutants with more than one type of mutagen. Point mutagensmay also prove useful in creating NOVA mutations in endogenous genesadjacent to EP insertion lines (Rørth et al (1998)), which carry UASsequences that can activate expression of neighboring genes in aGAL4-dependent fashion.

The ability of transposase to target rearrangements to sequences withinP elements has been exploited previously to induce loss-of-functionmutations in a hs-fused transgene (The'rond et al (1996)). An importantdifference between this earlier screen and the NOVA mutagenesisdescribed here is the use of a high-level expression system, whichInventors believe is critical for the recovery of novel activities thatwould otherwise go undetected. For example, the two noveldominant-negative Egf-r alleles and the dominant-negative Star^(DN) arevery dosage sensitive. The requirement for high-level expression ofdominant-negative constructs presumably reflects the need to produce asignificant excess of the mutant protein relative to the endogenousprotein to titrate out a sufficiently large fraction of the proteins orfactors interacting with the wild-type protein. However, these highexpression levels may also occasionally result in artifactual phenotypescaused by low-affinity interactions between proteins that would notordinarily occur to a significant extent. It is therefore important toconduct additional experiments (e.g., with loss-of-function mutants,deficiencies, or duplications) to confirm the biological relevance ofeach NOVA phenotype.

The results disclosed hereinabove suggest that the NOVA method could beemployed to generate dominant alleles of a wide range of genes usingvarious mutagens and provide insight to the function and mechanism ofaction of novel genes. This approach should be of particular utility ininvestigating the function of human disease genes, which have no knownfunctional motifs but have homologues in Drosophila (Reiter et al(2001)). Furthermore, NOVA mutagenesis should be applicable to anyorganism in which it is possible to misexpress transgenic constructs athigh levels in a conditional fashion.

The abovementioned examples define a new method, the NOVA Method, thatshould be applicable to any system in which overexpression/misexpressionis possible. The two specific examples that might be the most relevantare plants and single cell assays (e.g. frog oocytes or tissue culturecells). In the case of plants, the 35S misexpression system (patent heldby Monsanto) or any tissue specific form of misexpression should make itpossible to misexpress a given gene of interest either globally (35S) orin particular cell types (e.g. using enhancers specifically active inflowers, leaves, fruits, or roots). One could then perform NOVA exactlyas described for Drosophila (e.g. mutagenize transgenic plants andscreen for new dominant phenotypes genetically). Alternatively, since itis possible to transform plants at high efficiency using Agrobacterium,it should be possible to mutagenize a gene of interest in vitro usingvarious PCR or targeted mutagenesis schemes, subclone the mutated poolof cDNAs into an expression vector (e.g. 35S), transform the mutatedpool of cDNAs into plants, and then screen for dominant phenotypes.

In the case of frog oocytes one could mutate a gene of interest in vitroin an RNA expression vector, and inject pools of RNA made from theseconstructs into oocytes and look for a phenotype that differs frominjection of the wild-type form of the gene. This could be a powerfulway to conduct a structure function analysis of an ion channel gene.Similar in vitro mutagenesis schemes followed by transfection into cellculture lines could be designed for generating NOVA alleles of a geneand then assaying for phenotypes in cell culture (e.g. growth, DNAsynthesis, DNA repair, altered cell morphology, altered expression ofregulated luciferase expression etc.)

EXAMPLE 9

Method for Creating Dominant-Negative Forms of Bacterial Toxins

There is an existing need in combating intracellular bacterial toxins,especially now that we live in fear of terrorism. A viable approach tothis problem is the creation of dominant-negative forms of bacterialtoxins using NOVA screens in Drosophila.

Antibiotics and vaccines can prevent further bacterial growth, but donot inhibit the effect of already released bacterial toxins, which candestroy tissue without continued bacterial growth. Dominant-Negativeversions of such toxins may in principle prevent their effects insidethe infected cells. Over-expression of bacterial toxins in transgenicDrosophila lines could be used to reproduce the toxin-host cellinteraction, and to screen for Dominant-Negative versions of thesetoxins. For example, FIG. 12 shows that many bacterial toxins modifyRho-like GTPases at specific residues, which are conserved in Rho/Racfly homologues. This invention could be used to counter these toxineffects as follows:

1) The first step is to create transgenic flies able to express abacterial toxin using the UAS/GAL4 conditional expression system. Thisis accomplished by preparing DNA encoding full-length bacterial toxin(e.g. from genomic clone, plasmid subclone, or by PCR), inserting thetoxin-encoding gene into the pUAS-vector (Brand and Perrimon),transforming into E. Coli DH5α, testing for successful recombinants,preparing and purifying the pUAS-toxin DNA, injecting the w+ markedpUAS-toxin construct into w− fly embryos, isolating w+ transformants inF1 generation, establishing balanced transformed pUAS-toxin lines,expressing UAS-toxin in specific tissues using a set of GAL lines togenerate viable phenotypes (e.g. wingless or eyeless phenotypes).

FIG. 13 is a basic scheme for creating Dominant-Negative alleles ofbacterial toxins. In most cases, a very strong phenotype or death, iscaused by two doses of wild-type toxin (e.g. wild-type toxin from femaleplus unmutated UAS-toxin* from male. In rare cases, a DN form of thetoxin (toxin) reduces the phenotype caused by wild-type toxin.

FIG. 14 is a variant genetic scheme using chemical mutagenesis. In mostcases, a very strong phenotype, or death, is caused by two doses of thewild-type toxin. In rare cases, the DN toxin counteracts the effect ofthe wild-type toxin: no or reduced phenotype.

2) The second step involves determining whether the Drosophila toxinphenotype is caused by the same mechanism as in human cells. This isdone by analyzing the phenotype at the cellular level in third instarlarvae. The procedure is as follows:

a. Analyze the phenotype at the cellular level in third instar larvaecytoskeleton (for toxins affecting Rho-like GTPases). Test thereorganization of the actin; determine the shape and size of the cells(for Cytolethal Distending Toxins); test the activity of MAPK usinganti-diphosphoMAPK (for LF of B. anthracis); test the activity of Adenylcyclase (for EF of B. anthracis); and test if the toxin inducescell-lethality, and whether it occurs through apoptosis or necrosis.

b. Use genetic epistatsis experiments to confirm that the cellulartargets of the toxin are fly homologues of the human targets:

Test if the toxin-induced phenotype is modified in a heterozygous mutantbackground for the predicted target, and for their known partners;screen for enhancers of the toxin induced; and when the cellular targetis not known, a phenotype analysis could be undertaken to identify suchtargets.

3) The third step is analyze the effect of the novel dominant-negativetoxin. The procedure is to establish a mutant line carrying theUAS-toxinDN; determine what effect the toxinDN has when expressed alone(it should have no effect, even when expressed at high levels);determine by PCR what lesion has been created in the UAS-toxininsertion, and the corresponding altered protein sequence; clone amutant toxinDN gene into a fresh pUAS vector; transform construct intoflies; express in toxinDN gene flies with GAL4; and confirm that this isthe only mutation required for DN toxin activity.

While the present invention has now been described in terms of certainpreferred embodiments, and exemplified with respect thereto, one skilledin the art will readily appreciate that various modifications, changes,omissions and substitutions may be made without departing from thespirit thereof. It is intended, therefore, that the present invention belimited solely by the scope of the following claims.

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1. A method for generating dominant-negative forms of bacterial toxins,comprising the steps of: a. misexpressing a wild-type bacterial toxingene of interest in Drosophila flies, using a GAL4/UAS expressionsystem; b. determining the phenotypic consequence of such misexpression,and selecting flies in which the bacterial toxin gene of interest isexpressed; c. mutagenizing the wild-type bacterial toxin gene in theselected flies to produce carriers of a mutant bacterial toxinDN genethat differs in structure from the wild-type toxin gene by i. preparingDNA encoding full-length bacterial toxin, ii. inserting thetoxin-encoding gene into the pUAS-vector iii. transforming into E. coliDH5ÿ, iv. testing for successful recombinants, v. purifying thepUAS-toxin DNA, vi. injecting the pUAS-toxin construct comprising amarked transgene into fly embryos without the marked transgene,isolating transformants comprising the marked transgene in F1 generationcarrying a mutant bacterial toxinDN, and vii. selecting and establishingbalanced transformed pUAS-toxinDN fly lines; and d. causing the mutantbacterial toxin gene to be expressed in the selected flies by expressingUAS-toxin in specific tissues using a set of GAL lines.
 2. The methodaccording to claim 1, further comprising analyzing the activity of thenovel dominant-negative toxin by a. establishing a mutant line carryingthe UAS-toxinDN, b. determining what effect the toxinDN has whenexpressed alone, c. determining by PCR if a lesion has been created inthe UAS-toxin insertion and the corresponding altered protein sequence,d. cloning a mutant toxinDN gene into a fresh pUAS vector to obtain aconstruct, e. transforming the construct into flies, f. expressing intoxinDN gene flies with GAL4; and g. confirming that the mutation is theonly mutation required for the toxinDN activity.
 3. The method accordingto claim 2, wherein the mutations involve lethal factor (LF) of B.anthracis, the method further comprising analyzing the activity of MAPKusing anti-diphosphoMAPK antibody.
 4. The method according to claim 2,wherein the mutations involve LF of B. anthracis, the method furthercomprising analyzing the activity of Adenyl cyclase in the presence ofthe toxinDN.
 5. The method according to claim 2, wherein the mutationsinvolve Cytolethal Distending Toxins, the method further comprisingobserving the reorganization of actin to determine the shape and size ofthe cells developed in the presence of the toxinDN.
 6. The methodaccording to claim 1, further comprising a step d′ of determiningwhether the Drosophila toxin phenotype is caused by the same mechanismas in human cells by: d′. analyzing the phenotype at the cellular levelin third instar larvae cytoskeleton for toxins affecting Rho-likeGTPases by testing if the toxin induces cell-lethality and whether itoccurs through apoptosis or necrosis.
 7. The method according to claim6, wherein the step d′ of determining whether the Drosophila toxinphenotype is caused by the same mechanism as in human cells isaccomplished with genetic epistatsis experiments.
 8. The methodaccording to claim 7, wherein the genetic epistatsis experimentsperformed in step d′ include the steps of: i. testing to determine ifthe toxin-induced phenotype is modified in a heterozygous mutantbackground for the predicted target and for their known partners, ii.screening for enhancers of the toxin induced, and iii. performing aphenotype analysis to identify such targets when the cellular target isnot known.